| Part 1. Optimal dose of apocynin, NADPH oxidase inhibitor, to protect severe acute pancreatitisObjective:Aim of this study is to investigate the optimal dose of apocynin to protect rats with severe acute pancreatitis (SAP) and provide the optimal dose of apocynin to treat SAP caused intestinal injury in rats.Methods:Fifty male Wistar rats were randomly divided into five groups(N=10 in each group):sham operating group(SO), severe acute pancreatitis (SAP) group, apocynin treatment group 25mg/kg(APO25mg/kg), apocynin treatment group 50mg/kg(APO50mg/kg) and apocynin treatment group 100mg/kg(APO100mg/kg). SAP was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Apocynin was administered via femoral vein 30 min prior to SAP induction in the APO treatment groups. All rats were sacrificed 12h subsequent to SAP induction. Blood samples were obtained for serum amylase(AMY), lipase(LIP), alanine aminotransferase(ALT) and creatinine(Cr) detection. The quantity of collected ascites were measured. Pancreatic tissue samples were harvested for pathological examination.Results:The levels of ascites, serum AMY, LIP, ALT, Cr and pancreatic pathological score were significantly increased in SAP group than those in SO group(P<0.05). There was no difference between SAP group and APO 25mg/kg group. The levels of ascites, serum AMY, LIP and pancreatic pathological score in APO 50mg/kg and APO100mg/kg group were significantly lower than SAP group(P<0.05), but there is no significant difference between those two groups. Serum levels ALT and Cr in APO100mg/kg group were significantly higher than APO 500mg/kg group (P<0.05).Conclusion:25mg/kg apocynin has limited effect on SAP rats and can not reduce the severity of SAP.50mg/kg and 100mg/kg apocynin can effectively decreased ascites, serum AMY, LIP and pancreatic pathological score, and exert strong protective effect on SAP. However, 100mg/kg apocynin has greater liver and kidney toxicity than other does groups.We conluded that apocynin reduced severity of SAP and 50mg/kg is the optimal dosage vital for that effect.Part 2. Protective effects of apocynin, NADPH oxidase inhibitor, on intestinal mucosal injury in a rat model of severe acute pancreatitisObjective:This study aimed to investigate the protective effects of apocynin on intestinal mucosal injury in a rat model of severe acute pancreatitis (SAP).Methods:Sixty male Wistar rats were randomly divided into four groups:sham operating group(SO)(N=10), severe acute pancreatitis (SAP) group(N=30), apocynin treatment (APO)group(N=10) and drug control(APO-CON) group(N=10). SAP group further devided to three subgruops at 3h,6h and 12h time points. SAP was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Apocynin was administered 30 min prior to SAP induction in the APO treatment groups. All rats were sacrificed according to their time points subsequent to SAP induction. Serum leves of amylase(AMY), lipase(LIP), DAO, D-lactate and inflammatory cytokines were measured. The mortality of rats was calculated. Intestinal MDA and SOD were detected. The morphological change of intestinal tissue was analyzed under the light and transmission electron microscopy.Results:The levels of mortality, serum AMY, LIP, DAO, D-lactate, inflammatory cytokines, MDA, pathological injuries and ultrastructural changes of intestinal tissue in SAP were significantly increased than SO group(P<0.05). However, pretreatment with apocynin effectively decreased all these parameters(P<0.05). There was no difference between SO group and APO-CON group(P>0.05).Conclusion:Apocynin plays proactive role on SAP-associated intestinal mucosal injury by decreasing mortality, pathological injury of intestine, reducing oxidative stress and limiting inflammatory cytokines production.Part 3. The mechanism of apocynin’s protective effects on intestinal mucosal injury in a rat with severe acute pancreatitisObjective:This study aimed to investigate the exact mechanism of how apocynin could protect SAP associated intestinal mucosal injury in rats.Methods:Sixty male Wistar rats were randomly divided into four groups:sham operating group(SO)(N=10), severe acute pancreatitis (SAP) group(N=30), apocynin treatment (APO)group(N=10) and drug control(APO-CON) group(N=10). SAP group further devided to three subgruops at 3h,6h and 12h time points. SAP was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Apocynin was administered 30 min prior to SAP induction in the APO treatment groups. All rats were sacrificed according to their time points subsequent to SAP induction. NOX2, P38MAPK, NF-κB, caspase3 and caspase 9 expression in the intestine were evaluated either by immunohistochemical staining and western blotting. Apoptotic rate of intestinal epithelial cells were evaluated by TUNEL staining.Results:intestinal expression of NOX2, P38MAPK, NF-κB, caspase3 and caspase 9 and intestinal apoptotic index were significantly increased in SAP group than SO group(P<0.05). All these were redused significantly after the apocynin pretreatment(P<0.05). There was no difference between SO group and APO-CON group(P>0.05).Conclusion:These results suggested that NADPH oxidase inhibitor apocynin attenuates intestinal barrier injury in SAP rats, presumably because of its role on prevent ROS generation and inhibits the activation of P38MAPK, NF-κB and caspase pathway. |
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